Iron deficiency is the most prevalent nutritional problem in our country today. Iron overload promoted by hereditary hemochromatosis is one of the most common genetic disorders in our population. Increased knowledge about iron transport factors and how they are regulated to protect against iron deficiency and overload is essential to address these significant health problems. We have applied the emerging technologies of chemical genetics to advance our understanding of the factors and mechanisms involved in different pathways of iron uptake to discover several new transport inhibitors. Our goals are to characterize the activity of these compounds in vivo and in vitro and to discover new small molecule inhibitors using chemical genetics. We will use these reagents to advance our understanding of the factors, mechanisms, and regulation of iron uptake through the following specific aims: 1) To characterize the influence of small molecule inhibitors on iron transport in rats and mice. Using chemical genetics, we discovered ferristatin inhibits iron uptake from receptor-mediated endocytosis of the serum iron-binding protein transferrin. Ferristatin also blocks iron uptake by DMT1, a ferrous iron transporter that functions in dietary iron absorption across the apical membrane of enterocytes and after iron delivery into endosomes by the transferrin-mediated pathway. We propose to examine the effects of ferristatin and related compounds in vivo; 2) To determine the molecular mechanism of ferristatin inhibition of iron transport in vitro. We have shown that ferristatin inhibits transferrin-mediated iron uptake by inducing degradation of transferrin receptors by a clathrin- independent endocytic pathway. The actions of ferristatin are antagonized by cholesterol depletion, suggesting that lipid raft microdomains may be involved. Ferristatin also blocks iron uptake by DMT1 but the mechanism of inhibition remains to be identified. We propose to test the hypothesis that ferristatin modulates both iron transport pathways through lipid microdomains using cell molecular approaches; and 3) To identify and characterize novel small molecule inhibitors of DMT1. Recent combinatorial library screens have yielded a new class of structurally-related small molecule inhibitors of DMT1. We propose to define their effects on DMT1-mediated transport, transferrin-mediated iron uptake and non-transferrin bound iron uptake and to continue the discovery of inhibitors using the Molecular Libraries Screening Network (MLSN) system. PUBLIC HEALTH RELEVANCE: Iron deficiency remains the most prevalent nutritional problem in our country, yet recent identification of the gene responsible for hereditary hemochromatosis indicates that 1 in 20 Caucasians carry the defective allele and thus 1 in 400 may be susceptible to iron overload. Increased knowledge about the transport factors and how they protect against iron deficiency and overload is essential to more broadly address these significant health problems.